1. Field of the Invention
The present invention broadly relates to a method and apparatus for conducting gel electrophoresis. The invention specifically relates to a method and apparatus for resolving a complex mixture of components into a fixed two-dimensional array of its constituents in a gel matrix. In its preferred form, the invention pertains to a method and apparatus for the separation of complex mixtures of bio-organic molecules such as proteins and nucleic acids on the basis of two independent physical characteristics.
2. Description of the Prior Art
At present, in order to obtain very high resolution in the separation of bio-organic molecules one generally must employ the technique of two-dimensional gel electrophoresis first described by P. H. O'Farrel, (1975) Journal of Biological Chemistry, 250:4007-4021). By utilizing this technique, exceedingly complex mixtures of bio-organic molecules, containing for example over 1,000 different proteins, are separated and analyzed at one time.
According to this delicate, labor-intensive and time-consuming procedure, components of a protein mixture are separated in a two-step procedure which first involves a partition of proteins as a function of net surface charge by isoelectric focusing (IEF) in one dimension, followed by separation as a function of size (by molecular sieving) in a second dimension.
This separation process is carried out in a jelly-like material called a polyacrylamide gel. This gel is cast by polymerization of a mixture of acrylamide monomer and an appropriate cross-linking agent, such as N,N,N',N'-tetramethylethylene-diamine (TEMED), in suitably sized glass tubes or between rectangular glass plates to form rods or thin sheets, respectively.
Isoelectric focusing (IEF), the first separation step, relies on the fact that bio-organic molecules such as proteins and peptides, are three-dimensional objects with ionizable surface groups (e.g., carboxyl, amino, imidazole, guanidinium, etc.). These ionizable groups are amphoteric in nature; i.e., below a certain pH, such groups are positively charged and above a certain pH, they are negatively charged. At a particular pH value, called the isoelectric point (pI), the number of positively charged surface groups equals the number of negatively charged surface groups on the molecule. Consequently, the molecule will have a net charge of zero.
IEF involves the electrophoretic migration of a molecule in a pH gradient until it reaches the pH corresponding to its isoelectric point. As the net charge of the molecule is zero at that point, it becomes immobilized and remains "focused" at its respective pI.
Stable pH gradients are generated within polyacrylamide gels by inclusion of specialized buffer constituents called ampholytes. Ampholytes consist of a mixture of low molecular weight amphoteric compounds with isoelectric points covering a defined range of pH values. Application of a constant-voltage, DC electric field to an ampholyte-buffered gel causes the ampholytes to focus according to their pIs, thereby establishing a pH gradient with sufficient buffering capacity and conductance for subsequent focusing of amphoteric macromolecules.
After performing the first dimension IEF separation in a gel, cast in a glass tube, the technician performing the separation then must carefully remove the gel rod, which contains the focused proteins, from the glass tubing.
Typically, each gel rod then is treated with a second dimension electrophoresis buffer solution. This aqueous buffer solution contains a detergent such as sodium dodecyl sulfate (SDS), which binds to proteins in the gel providing these macromolecules with a net negative charge. Afterwards, each gel sample is placed on top of and cemented to one edge of a second dimension polyacrylamide gel slab.
The assembled composite consisting of the treated first dimension gel overlaying and secured to the second dimension gel slab then is placed within an electric field between appropriate electrically conductive buffer solutions and separation in the second dimension may proceed on the basis of the proteins' differing molecular sizes via the process of molecular sieving.
Depending upon the particular apparatus design and gel and buffer chemistries, the technique of two-dimensional gel electrophoresis requires anywhere from about 17 hours to more typically about 30 hours to perform each separation. Moreover, not only is this separation procedure quite labor-intensive and time-consuming, but as those skilled in this technology readily appreciate the procedure also demands very high expertise and exacting laboratory techniques on behalf of each operator to ensure reproducibility of results. Unavoidably, the technique also places the fragile polyacrylamide gel containing the separated components generated in the first dimension at considerable risk during the transfer process preparatory to the second dimension separation.
As a consequence of these substantial drawbacks, two-dimensional gel electrophoresis has not been used for many applications where the procedure is ideally suited, such as for example clinical diagnostic screening and other large scale analytical applications.
Another separation technique useful for separating bio-organic molecules which continues to receive considerable attention from both the basic researcher and clinical diagnostician is high performance liquid chromatography (HPLC). Although incapable of achieving the same degree of resolution possible with two-dimension gel electrophoresis, this procedure nevertheless provides a very high resolution of the components in a complex mixture. In fact, recent advances in this separation technology have virtually eliminated the gap between this technique and one-dimensional electrophoresis techniques. More importantly, when using this technology the separation of complex mixtures of bio-organic molecules is achieved in only a fraction of the time necessary to carry out the process of gel electrophoresis.
According to this technique, a mobile phase or eluate, into which the sample to be analyzed has been injected, is forced through a bed of microparticulate chromatographic packing material at a relatively high linear velocity. Velocities on the order of 0.1 millimeter/second or greater are typical. As recognized by those skilled in this art, separation of components in the sample depends in large part upon eluate chemistry and the nature of the packing material used. Proteins and other bio-organic molecules are separated in this procedure, inter alia, on the basis of size (gel permeation chromatography), ionic properties (ion-exchange), absorptive characteristics (absorption chromatography) and hydrophobicity (reversed phase chromatography).
Analysis of the separated components typically is accomplished using in-line detectors measuring such component properties as absorbance and refractive index. It also is known to couple the detector and the chromotagraphic column through a post-column reactor wherein separated components of the sample are converted, by reaction with appropriate reagents, into fluorescent or color derivatives. The so-altered species then can be identified using an appropriate detector.
One object of the present invention is to provide a method and apparatus for automating gel slab electrophoresis.
Another object of the present invention is to provide a method and apparatus suitable for sequentially resolving a complex mixture of components and particularly a mixture of bio-organic molecules, preferably according to two independent physical characteristics.
A further object of this invention is to provide a method and apparatus for separating a complex mixture of components, particularly a mixture of bio-organic molecules, which method and apparatus exhibit advantages of both high performance liquid chromatography and two dimensional gel electrophoresis techniques.
Yet a further object of this invention is to provide method and apparatus which automate the sequential separation of a complex mixture of components, particularly a mixture of bio-organic molecules, that has heretofore been accomplished using the well-known manual procedure of two-dimensional gel electrophoresis, without losing the resolving power inherent in such well-known technique.
These and other objects of this invention will become apparent from a consideration of the specification and appended claims.